Glutamate neurotoxicity has been proposed as a common secondary pathway to cell death in many neurodegenerative diseases, including Amyotrophic Lateral Sclerosis (ALS). In support of this hypothesis, recent evidence demonstrates that in the spinal cord, brainstem and motor cortex of roughly 65% of patients with sporadic forms of ALS, protein levels for the EAAT2 sub-type of glutamate transporter are greatly reduced. These changes in EAAT2 protein levels are correlated with aberrant splicing of the EAAT2 mRNA, resulting in proteins that either undergo rapid degradation and/or produce a dominant negative effect on normal EAAT2 trafficking. The net result of these changes is a reduction in glutamate uptake and a mechanism for increasing extracellular glutamate concentrations to neurotoxic levels. To address the role of glutamate transport in excitotoxic cell-death, we have recently generated a transgenic model of EAAT2 overexpression. Transgene expression is driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter and results in a 3-5 fold increase in synaptosomal, hippocampal, cerebrocortical and spinal cord D-aspartate uptake. In a kainic acid model of excitotoxic cell death, EAAT2 transgenic mice demonstrate a significant decrease in hippocampal cell death, which correlates with attenuation of the c-Jun immediate early gene stress response pathway. In transgenic models of motor neuron degeneration, EAAT2 protein levels are also diminished at either disease onset and/or end stage disease. Therefore, to test the hypothesis that excitotoxicity contributes to motor neuron cell death in these models, we have crossed EAAT2 transgenic mice with SOD1 G93A transgenics. Disease onset in the double-transgenic mice is delayed by 30 days, compared with clinical onset in SOD1 G93A littermates. The purpose of this grant is to further test the hypothesis that excitotoxicity is a component of motor neurodegeneration through the characterization of EAAT2/SOD1 G93A double transgenics. Using a non-SOD1 model of motor neuron loss, which also exhibits decreased glutamate uptake with disease onset, we will test the hypothesis that enhanced glutamate uptake plays a neuroprotective role in chronic neurodegenerative disease processes. To investigate mechanisms that lead to, or contribute to, motor neuron cell death, we propose to study the roles of astrocytic differentiation and gliosis in the regulation of EAAT2 mRNA and protein levels, and EAAT2 cellular distribution in spinal cord organotypic and primary culture.